Cardiomyocytes have a finite life-span in culture before becoming terminally differentiated. Attempts to immortalize ventricular cardiomyocytes in order to establish a cell line that can proliferate in culture have not been successful. Recently, Claycomb et al (1998) described a mouse atrial cardiomyocyte cell line, HL-1, which can be passaged serially, differentiate, and maintain the characteristics of adult mouse cardiomyocytes. This cell line could be a useful tool for cardiovascular research, but a mouse cell line can not answer questions that are specific to the human system. Currently there is no immortalized human cardiomyocyte cell line that can proliferate and differentiate in culture and can express the adult cardiomyocyte phenotype when culture conditions are manipulated. Using an unexpected, novel and unique mitochondrial function-based method to immortalize human primary cardiomyocytes from both adult and fetal heart tissue the cell lines described in this report have been passaged for over 100 generations and can be regrown from frozen stocks. The cells express both cardiac and skeletal muscle-specific markers and can be induced to differentiate in culture to express adult skeletal muscle and cardiac muscle phenotypes.